The Role Of Key Molecules Of Non-neuronal Cholinergic System In EMT Of A549 Cells Induced By Coal Reference Materials

Objective To explore the role of key molecules of the non-neuronal cholinergic system in the epithelial-mesenchymal transition(EMT)of human lung adenocarcinoma A549 cells induced by coal reference materials.Methods The A549 cells were exposed to coal reference materials at the final concentration of 0,1,2,4,8,16 and 32μg/cm~2for 24 h.CCK8 assay was used to detect the effect of A549 cells on cell proliferation under exposure conditions of each dose group.A549cells were exposed to 0,1,2,4 and 8μg/cm~2of coal reference materials.After exposure for24 h,the expression levels of EMT marker protein Vimentin and E-cadherin were detected by Western blot.Enzyme-linked immunoassay(ELISA)was used to determine the contents of interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α).The expression levels of superoxide dismutase(SOD)and malondialdehyde(MDA)were determined by colorimetry.The change of acetylcholinesterase(ACh E)activity was determined by modified Ellman method.The m RNA relative expression levels of acetylcholine receptors CHRNB2,CHRM3,CHRM5,CHRNA5,CHRNA7 and CHRNA9 were detected by quantitative real-time polymerase chain reactions(q RT-PCR).Exogenous ACh E recombinant protein with the final concentration of 0,0.2,1 and 5 U/m L was exposed to A549 cells for 24 h,and the effect of each dose group on the proliferation activity of A549 cells was determined by CCK8 method.A549 cells were exposed to ACh E with final concentration of 0,0.2,1,5 U/m L and 4μg/cm~2coal reference materials for 24 h.The contents of IL-6,TNF-α,SOD,MDA and m RNA relative expression levels of acetylcholine receptors CHRNB2,CHRM3,CHRM5,CHRNA5,CHRNA7,and CHRNA9 were determined by the above methods.Results 1.After treated with different doses of coal reference materials for 24h,the proliferation activity of A549 cells were decreased gradually with the increase of exposure dose.When the exposure dose of coal reference substance reached 4 and 8μg/cm~2,the protein expression level of Vimentin was significantly increased,while the protein expression level of E-cadherin was significantly decreased,with statistical significance(P<0.05).With the increase of coal reference materials exposure dose,SOD showed a gradually decreasing trend,while IL-6,TNF-αand MDA showed an increasing trend,and ACh E enzyme activity showed a gradually decreasing trend,the difference was statistically significant(P<0.05).The m RNA relative expressions of acetylcholine receptor CHRM3 and CHRNA9 in A549cells were increased gradually,and the m RNA relative expressions of CHRM5,CHRNA5 and CHRNA7 were decreased gradually(P<0.05),while the m RNA relative expression of CHRNB2 had no significant change.2.The proliferation activity of A549 cells was not significantly changed after treatment with different doses of exogenous ACh E recombinant protein for 24 h.Coal reference materials and exogenous ACh E recombinant protein interaction in A549 cells for 24 h,with the increase of ACh E exposure dose,compared with single exposure coal group standard substance,to a certain extent,inhibiting A549 cell SOD and IL-6,TNF-α,the rise of MDA,when reaching 4,8μg/cm~2,the differences were statistically significant(P<0.05).At the same time,it can also restore the down-regulation phenomenon of m RNA expression levels of acetylcholine receptors CHRM5,CHRNA5 and CHRNA7 in A549 cells caused by coal reference materials.Conclusion The high dose of coal reference materials was cytotoxic to A549 cells.A certain dose of coal reference materials can promote the occurrence and development of EMT in A549 cells,resulting in inflammatory response and oxidative stress in A549 cells.At the same time,it can reduce the activity of ACh E enzyme and affect the relative expression level of acetylcholine receptor m RNA,while the addition of exogenous ACh E recombinant protein can inhibit this phenomenon to a certain extent.These results indicate that key molecules of non-neuronal cholinergic system play a certain role in EMT induced by coal reference material in A549 cells,but the specific mechanism needs to be further explored in the future.